Compositions and methods for peptide-assisted transfection

ABSTRACT

Compositions that increase the efficiency of nucleic acid transfection of cells are provided, including peptide transfection reagents and fusion proteins containing the peptides. Also provided are methods of using the peptide transfection reagents and fusion proteins to transfect cells.

This application claims the benefit of priority under 35 U.S.C. § 119 ofU.S. Ser. No. 60/484,394, filed Jul. 1, 2003, the entire content ofwhich is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to peptides that have a desirablenon-specific dsDNA binding ability and are useful for facilitatingtransfection of eukaryotic cells. Also disclosed are variouscompositions and methods of transfecting eukaryotic cells utilizing suchpeptides. The invention also relates to compositions and methods ofcombining peptides with salt and known transfection reagents fortransfection.

2. Background Information

Delivery of genetic material into cells is important in many areas ofresearch, including, for example, studies of gene function, geneticpathway analysis, examination of cell morphology in relation to geneticalteration, and cell-based library screening. Delivery of geneticmaterial into cells is also a critical step of gene therapy, which canprovide benefit to an organism. Transfection is the standard method bywhich cells can be induced to take-up nucleic acids and producepolypeptides useful as production units for manufacturing or asscaffolds for tissue engineering (for review, see [Godbey, 2001 #150],the full citation for all references cited herein are listed can befound following the Examples, below). Introduction of the cDNA of genesthat play roles in lineage maintenance or commitment can facilitate theuse of stem cells for cell-based therapy [Reya, 2003 #151].

Most high efficiency gene deliveries, especially those in clinicalstudies, rely on viral vectors as a carrier, for example, lentiviral andadenoviral vectors. Factors influencing viral vector usage includelengthy procedures of virus preparations, genome packaging limits,toxicity and safety, and target cell tropism. In many in vitro and exvivo cases, non-viral gene delivery systems is selected because of itssimplicity of use. Genetic material can be delivered by transfectionwith various techniques, including, for example, calcium precipitation,complexation with cationic lipid, DEAE-dextran, dendrimer polymers,polybrene or other cationic reagents. Delivery can also be mediated byelectroporation or a mechanical method such as microinjection orbiolistic methods, which can be particle based.

Liposomes and cationic polymers currently make up the two major classesof chemical gene delivery methods. Liposome-mediated transfectionprovides advantages such as relatively high efficiency in a wide varietyof cell types, ability for delivery of DNA of all sizes ranging fromoligonucleotides to yeast artificial chromosomes [Lamb, 1995 #152],delivery of RNA [Malone, 1989 #153], and delivery of proteins [Debs,1990 #156]. DNA transfected into cells by liposomes can be integratedinto chromosomes for long-term experiments. There are an increasingnumber of studies that use liposomes for delivery of nucleic acid toanimals and humans [Felgner, 1995 #157][Liu, 1995 #155; Liu, 1997 #154].However, a major problem with liposome-mediated transfection is thatintracellular accumulation of secreted proteoglycans compete with DNA toform complexes with liposome and thereby reduce transfection efficiency[Belting, 1999 #21].

One of the most successful polycations used as transfection reagent ispoly(ethylenimine) (PEI). Compared to common liposomes, PEI seems toprovide better protection of DNA against nuclease digestion [Ferrari,1999 #158], while demonstrating equal or better transfection capabilityin vitro and in vivo. The ability of PEI to compact DNA is believed tobe responsible for keeping large cargo DNA molecules (e.g., yeastartificial chromosomes of 2.3 Mb) intact and delivering them intoeukaryotic cells [Marschall, 1999 #159]. Unfortunately, a disadvantageof using PEI, as well as several other transfection reagents, is thehigh level of toxicity to cells exposed to either free or DNA-complexedPEI [Godbey, 2001 #29].

Due to the disadvantages associated with existing transfection system,many other molecules are being tested for gene delivery. Most of theseexperimental reagents and methods utilize a form of cationic molecule informing complexes, at physiological pH, with negatively charged DNAmolecules, and bringing the DNA into close contact with the negativelycharged cell surface. Examples of such reagents include chitosan[Richardson, 1999 #160], and β-cyclodextrin [Gonzalez, 1999 #161].Various degrees of success have been reported in comparison with thoseusing CaPO4, LIPOFECTAMINE transfection reagent, PEI, and other“classic” reagents.

Regardless of the method chosen for transfecting a particular type ofcells, the following obstacles must be overcome for an effectiveexpression of introduced DNA: 1) close contact of DNA-containing complexwith cell surface, 2) passage through cell membrane, 3) dismantling ofthe DNA-encapsulating complexes in the cytoplasm in most cases, and 4)nuclear import of the DNA. The mechanism of cell entry remains unclearfor most established transfection methods, although endocytosis islikely a main route for DNA complexes to enter cells. The fate ofdifferent DNA-containing complexes, once inside cytoplasm, depends onthe type of reagents used to precipitate or surround the DNA molecules.The mechanism for the degradation or dismantling of the complexes alsois not fully understood. A tight DNA-encompassing complex may befavorable in surviving the process of contacting and penetrating cellsurface, but may not be efficient in releasing DNA for nuclear entranceand gene expression. In addition, reagents that bind DNA often can bindserum components as well, resulting in less transfection in the presenceof serum in cell medium or in vivo. An example is the use of cationiclipid oleoyl-ornithate (OLON) in combination withdioleoylphosphatidylethanolamine (DOPE), which gives a highertransfection efficiency than other liposomes, presumably because ofthermodynamically more favorable separation of the cargo DNA moleculesafter cellular entry [Tang, 1999 #162]. As another example,phosphorothioate oligonucleotides delivered by PEI displayed higherantisense activity than similarly delivered phosphodiesteroligonucleotides at least in part due to more favorable thermodynamicsof releasing carried DNA [Dheur, 1999 #163; Dheur, 2000 #164].

Nuclear entry is another barrier for transgenic expression. By mosttransfection methods, the best chance for cytoplasmically located DNA toenter the nucleus is during the reorganization of the nuclear membraneduring cell division. In non-mitotic cells, nuclear envelop crossing byplasmid DNA is a very rare event [Escriou, 2001 #133]. Accordingly,transfection efficiency with polycation-based or lipid-basedtransfection systems could be 30-fold to 500-fold higher whentransfection is performed during S or G2 phase as compared with cells inG1 phase [Brunner, 2000 #131]. This factor can create a dilemma becausemany commercially available transfection reagents require the cells benear confluent at the time of transfection, leaving little room forsignificant cell division to occur following transfection. In addition,it is constantly observed that cells are temporarily arrested, due tothe cytotoxicity of most lipid-based or polycation-based reagents, andshow less cell division in a given time compared to untreated controls.The arrested cells often recover after growing in reagent-free mediumfor a day or two. Recent advances in improving nuclear transfer oftransfected DNA include the addition of nuclear localization signal(NLS), for instance, a signal peptide derived from SV40, to theDNA/liposome complex [Aronsohn, 1998 #165].

Peptides also can be included in a DNA carrier complex to enhance cellbinding through receptor recognition, thereby enhancing transfection.Certain viral proteins or their fragments increase the percentage oftransfected cells when included in cationic lipid-based transfectioncomplex (see, for example, Wickham et al. [Wickham, 1995 #166],Yoshimura et al. [Yoshimura, 1993 #167], Kamata et al. [Kamata, 1994#168], and Remy et al. [Remy, 1995 #169]). Hawley-Nelson et al. (U.S.Pat. Nos. 5,736,392; 6,051,429; and 6,376,248; and U.S. Pat. Appl. Publ.No. 2003/0069173) described a related system, which was stated to bedifferent than the above cited examples because it can significantlyimprove the efficiency of transfection when peptide is bound to nucleicacid prior to adding the transfection reagent. Hawley-Nelson et al. alsostate that peptides covalently coupled to the transfection agent, e.g.,directly or indirectly linked to a lipid or to a dendrimer, function toimprove transfection. It is not clear, however, how the suggested cellsurface-binding peptides such as VSVG and RGD can function similarlyeither pre-bound to DNA or linked to lipid, and how this system differsmethodologically from previous protocols. It appears that the effect ofthe peptides was solely due to the effect of polycation peptides bindingto DNA, as the addition of a polycationic peptide without NLS improvedtransfection even more than that of a cationic NLS. However, it is notclear how these DNA-bound positively charged peptides can also functionas NLS or other functional motif during complex transport.

There have been more recent successes in using peptides to directDNA-containing complexes to the surface of selected cells. Scott et al.synthesized a peptide containing an integrin-binding tripeptide (RGD)and a DNA-binding polylysine for enhancement of liposome-mediated genedelivery [Scott, 2001 #171]. Increased transfection was seen withintegrin-expressing cells, and the effect of the RGD peptide on cellcontact appeared specific as control RGE or competitive antibodiesagainst integrin reduced gene transfer. By a biotin-streptavidinmediated conjugation scheme, Lee et al. linked PEGylated epidermalgrowth factor (EGF) to PEI-DNA complex and achieved EGFreceptor-mediated endocytosis [Lee, 2002 #172].

There have also been examples of using nuclear location signals insimilar manner. Gopal et al. (U.S. Pat. No. 5,670,347) suggested apeptide composed of an NLS, a flexible hinge, and a basic DNA-bindingmotif; Gerhard et al (DE-OS 195 41 679) described nuclear localizationsignal- (NLS-) polylysine conjugate for gene transfer; and Szoka (PCT1993) bound an NLS to DNA via an intercalating chemical agent; Toth etal. used a lipophilic palmitoyl-peptide derived from SV40 T antigen NLSin combination with PEI [Toth, 2002 #49]. In all these cases,DNA-binding is intended to rely on either positively charged peptides orchemical moieties. In either case, it is plausible to expect thatcationic NLS binds DNA as well and may not function to their fullpotential as signal peptide. DNA-binding solely depending on positivecharge cannot survive competition by other negatively charged moleculesduring the process of transfection. In addition, the use of mutagenicintercalators described by Hawley-Nelson et al. limits the applicationof the methods.

For improvement over these methods, Siebenkotten et al. (U.S. Pat. No.6,521,456) described a system in which the main modifications was to usea specific DNA binding domain such as a lac repressor or a PNA moleculeas the DNA binding module, and a neutralized NLS as the targetingmodule. A drawback of this method is that for sequence-specificDNA-binding domains, a specific site (or sites) must be engineered intothe plasmid, thus limiting a broad use of the reagent. For PNA, aspecific PNA-peptide conjugate has to be made for each type of plasmid.Also, there can be no more than a few sequence-specific binding events,by definition, between each DNA and the transfection reagent molecules.Therefore, it is difficult to compact large DNA or to improvetransfection efficiency by increase the ratio of peptide/DNA. Eventhough Siebenkotten et al. disproved the earlier reports of usingproteins for transfection, e.g., HMG-1 by Kaneda (U.S. Pat. No.5,631,237) and thymus histones by Fritz et al. [Fritz, 1996 #170],partly for the reason of high cost and labor of producing proteinscompared to peptides, the lac repressor they used is a protein.

More recently, Behr et al. (U.S. Pat. Appl. Publ. No. 2003/0100113)described the use of a nuclear localization signal (NLS) covalentlylinked to an oligonucleotide, and noted that the NLS conjugate can becovalently linked to one or both termini of a linear DNA molecule,associated with a plasmid DNA molecule by forming a triple helix, orinserted in a plasmid DNA molecule by strand invasion. The transfectionvector is useful for gene therapy applications. This system is based ona peptide-oligonucleotide hybrid molecule, which requires specific siteon the delivered DNA to match with the oligonucleotide sequence on thehybrid, and chemical conjugation of oligo to peptide. As such, thesystem is not a very convenient and, therefore, is not likely to bewidely used. As a result of these limitations, there is great need toidentify materials and methods for gene delivery that can result in highefficiency, low toxicity, low cost, and convenience in broadapplications.

SUMMARY OF THE INVENTION

The present invention provides peptide transfection reagents,compositions that include such peptide transfection reagents, kitscontaining the transfection reagents and/or compositions, and methods ofusing the peptide transfection reagents and/or compositions fortransfecting a eukaryotic cell with high efficiency. Accordingly, thepresent invention relates to a peptide transfection reagent having theamino acid sequence QRNPNKKWS (SEQ ID NO:1), which is a peptide fragmentof a Nun polypeptide (“Nuc”). In one embodiment, the peptidetransfection reagent is a component of a fusion protein, which, inaddition to the peptide of SEQ ID NO:1, contains one or moreheterologous polypeptides operatively linked thereto In anotherembodiment, the peptide transfection and one or more heterologouspolypeptides are associated via a non-covalent interaction that isstable under physiological conditions, including conditions suitable forperforming a transfection reaction.

In one aspect, the heterologous peptide is a cellular localizationdomain, for example, a nuclear localization signal (e.g., PKKIKTED; SEQID NO:4), which facilitates transport of the fusion protein, and anynucleic acid molecule complexed thereto, into the nucleus of aeukaryotic cell. In other aspects, the cellular localization signalcomprises an HIV TAT peptide, for example, a TAT peptide having an aminoacid sequence including YGRKKRRQRRR (SEQ ID NO:2), which facilitatestranslocation of the heterologous polypeptide, and any nucleic acidmolecule complexed thereto, across a eukaryotic cell membrane and into acell, or is an HIV gp41 peptide GALFGGFLGAAGSTMGA; SEQ ID NO:5). Instill other aspects, the heterologous peptide can comprise a functionalsequence such as an endoprotease recognition site (e.g., a cathepsin Drecognition sequence; GGFLGF; SEQ ID NO:6), whereby, when a complexcomprising a nucleic acid molecule and a peptide comprising theendoprotease recognition sequence is localized in a region of a targetcell containing the endoprotease (e.g., a endosome, or cytosol),proteolytic cleavage occurs and some or all of the peptide is removedfrom the nucleic acid molecule. In another example, the functionalheterologous peptide can be an endosomolytic peptide such as theinfluenza virus fusogenic peptide, INF7 (GLFEAIEGFIENGWEGMIDGWYG; SEQ IDNO:7). In another embodiment, the peptide-nucleic acid complex caninclude an endosomolytic agent such as chloroquine operativelyassociated with the complex.

The peptide transfection reagent and one or more heterologouspolypeptide (e.g., an HIV TAT peptide (SEQ ID NO:2) or an HIV gp41peptide (SEQ ID NO:5, or a nuclear localization signal (SEQ ID NO:4),alone or in combination with an HIV TAT peptide or an HIV gp41 peptide)can be operatively linked by directly linking the peptides together, forexample, by forming a peptide (or other bond) between the C-terminus ofone peptide and the N-terminus of the second (or more) peptide(s), or byexpressing the fusion protein from a recombinant nucleic acid moleculeencoding the peptide components, in frame, or the peptide transfectionreagent and heterologous polypeptide(s) can be operatively linked via aspacer, which can be any molecule useful for linking two or morepeptides to each other, for example, an amino acid or peptide linker. Afusion protein of the invention is exemplified by SEQ ID NO:1operatively linked to SEQ ID NO:2 via a single glycine linker (see SEQID NO:3).

A composition of the invention can further include a nucleic acidmolecule, which, upon contact with the peptide transfection reagent,forms or is capable of forming a complex. The nucleic acid molecule canbe a single stranded or a double stranded nucleic acid molecule, and canbe DNA or RNA or a DNA/RNA hybrid. In addition, a composition of theinvention can include divalent cations, for example, divalent calciumions.

The present invention also provide a kit, which contains at least apeptide transfection reagent of the invention, and can further containreagents useful for performing and/or monitoring a transfection reactionand/or instructions for using the peptide transfection reagent fortransfecting a cell. As such, the kit can further contain one or aplurality of heterologous polypeptides, for example, a cellularlocalization domain or a plurality of different cellular localizationdomains. Preferably, the heterologous polypeptides are in a form thatfacilitates an association or operative linkage with the peptidetransfection reagent, for example, by having sequences that facilitatean association that is stable under physiological conditions or thatfacilitate the formation of a covalent linkage to the peptidetransfection reagent such that the heterologous polypeptide and peptidetransfection reagent each maintains its desired function. If desired, akit of the invention can further contain one or more reagents foroperatively linking the relevant peptides, either directly or via alinker moiety. Where the kit contains a plurality of such heterologouspolypeptides, an advantage is provided in that a user of the kit canselect an heterologous polypeptide as desired, i.e., depending on theparticular needs of the user.

The kit also can contain one or more reagents useful for performing atransfection reaction, including, for example, buffers, transfectablenucleic acid molecules useful a standard (controls) for monitoringtransfection efficiency, and the like. In one aspect, the kit containsdivalent calcium ions, either in a solution or in a form that can beplaced into solution.

The present invention further relates to a method of transfecting acell. In one embodiment, the method is performed, for example, bycontacting the cell, generally a eukaryotic cell (e.g., a mammalian cellsuch as a human cell) with a peptide transfection reagent as disclosedherein (e.g., SEQ ID NO:1) and a nucleic acid molecule under conditionssufficient from cell transfection. Such conditions can include, forexample, an appropriate concentration of divalent calcium ions. Inanother embodiment, the methods is performed, for example, by contactingthe cell with a fusion protein, which includes a peptide transfectionreagent operatively linked to a heterologous polypeptide (e.g., a fusionprotein as set forth in SEQ ID NO:3), and a nucleic acid molecule underconditions sufficient for cell transfection. Such conditions caninclude, for example, an appropriate concentration of divalent calciumions. In still another embodiment, the method is adapted to a highthroughput format, whereby, due to the high transfection efficiencyobtained using the disclosed compositions, a plurality of cells, whichcan be the same or different, can be transfected in parallel with one ormore polynucleotides (e.g., polynucleotides encoding small interferingRNA molecules), which can be the same or different.

DETAILED DESCRIPTION OF THE INVENTION

Methods and compositions are provided for delivering genetic materialinto cells by the function of a short peptide, Nuc, which can bind todouble stranded (ds) DNA and dsRNA in a manner that is not substantiallydue to an electrostatic interaction. As disclosed herein, linkage of theNuc peptide to a transmembrane domain peptide (e.g., HIV TAT) enhancestransfection mediated by other transfection reagents. As furtherdisclosed herein, the Nuc peptide can form complexes with DNA moleculesand, therefore, mediate transfection, alone. Unexpectedly, calcium ionstabilized the complex of DNA and Nuc-containing peptide. In combinationwith calcium ion, but not necessarily calcium phosphate precipitate, theNuc peptide resulted in transfection of DNA with efficiency higher than,or at least comparable to, commercial transfection reagents underpractically the best conditions. The use of the Nuc-containing peptidedid not result in any observable cytotoxicity, required a very lowamount of DNA and peptide, and was of significantly lower cost thanexisting lipid-based and polycation-based transfection reagents.

The present invention relates to a protein motif (Nuc), which is derivedfrom Nun, a natural DNA and RNA binding protein of phage HK022 (seeGenBank Acc. No. P18683; see, also, GenBank Acc. No. X16093, each ofwhich is incorporated herein by references). Nuc can functionindependently as a peptide that binds to dsDNA non-sequence-specificallywith affinities desirable for carrying nucleic acids into cells andallowing the genetic material to be expressed in the nucleus. Theinvention also relates to peptides further derived from the coresequence, QRNPNKKWS (SEQ ID NO:1), of the Nuc peptide. For example,peptides that have more than one repeat of the core sequence in a linearor branched form, peptides with limited residue substitutions, peptidesthat link the core sequence(s) to other functional motifs such as to aprotein transduction domain (PTD), other fusagenic peptides, a nuclearlocalization signal (NLS), receptor or surface protein binding domain,and charged peptides such polyarginine or polylysine. Derived peptidesalso can include those that have certain modified amino acids such asamino acids that are partially deprotected after chemical synthesis, orunnatural (non-naturally occurring) L-amino acids. Further, one or afew, e.g., 2, 3, 4, 5, or 6, amino acids can be linked to one or bothends of the Nuc peptide to provide a desired characteristic (e.g., acysteine residue can be linked via a peptide bond to a terminus of theNuc peptide (SEQ ID NO:1) to facilitate cross-linking of the Nuc peptidewith another Nuc peptide or other peptide of interest). The inventionalso relates to modifications that result in additional intercalating ofthe peptide to dsDNA or dsRNA, for example, a modification including oneor more added aromatic amino acids.

Prior to the present invention, one main family of DNA-binding peptideswas reported in the literature in regard to DNA transfection. These DNAbinding peptides were based on having positively charged amino acidsoften spaced in viral protein transduction domain (PTD). Such examplesinclude peptide JTS1 or GALA [Rittner, 2002 #56] and related KALA[Wyman, 1997 #57], CL-22 [Haines, 2001 #17]. Because the most commonlyused viral PTDs contain numerous positively charged amino acids, and areoverall basic, there were a few reports of using them directly as atransfection vehicle. For instance, HIV TAT [Sandgren, 2002 #4] and HIVVPR [Coeytaux, 2003 #174] has been reported to carry DNA into cells,although many of these studies only showed indirect measurement of thetransfection events. To the other extreme, simple repeats of cationicamino acids such as polyarginine have been tried for similar purposes[Kim, 2003 #1]. These peptides have two fundamental shortcomings: first,they are pH-dependent as protonation status influences the mainlyelectrostatic interactions between the charged amino acids and DNA; andsecond, these peptides do not distinguish dsDNA from ssDNA, RNA, certainserum proteins, polyanionic heparan sulfate proteoglycans or othercharged molecules in general [Sandgren, 2002 #4], making them somewhatnonspecific carriers that can deliver unintended cargo molecules intocells or animals.

It was therefore desirable to find other types of short peptide domainsthat specifically recognize dsDNA (and dsRNA) in a sequence-nonspecificmanner, and particularly with a medium range of affinity. Since dsDNA isthe form of transgene in most cases, it can be beneficial to havebinding biased toward dsDNA, thus minimizing, if desired, transfectionof ssDNA and most RNA. Recent development in the RNA interference (RNAi)field has illuminated the need to transfect dsRNA into cells. It istherefore also desirable to have a peptide that also can bind dsRNA bynon-electrostatic means. Binding in a sequence-independent manner canavoid the need for including a specific site as in the method ofSiebenkotten et al (U.S. Pat. No. 6,521,456), and can allow multiplebinding such that a stable complex can be formed and the DNA compactedfor transmembrane movement and protected from nuclease attacks. Amoderate binding affinity provides the greatest likelihood of creating atight enough complex between transfection agent and DNA fortransfection, and of releasing the DNA once inside the intendedlocation, generally the nucleus, for gene expression.

In an effort to identify a short peptide that binds dsDNA by mainly anon-static interaction, the C-terminal 9 amino acid region of phageHK022 Nun protein was found to meet the specified requirements [Watnick,2000 #44]. Extensively studied, this region interacts directly withdsDNA. Nun does not arrest polymerase on ssDNA template, indicating thatits DNA binding motif only binds dsDNA. Point mutations in theC-terminal region have illustrated its role for dsDNA binding andpinpointed the penultimate residue, tryptophan (W 108), as the mostimportant residue for binding. The fact that W108 can be functionallyreplaced only by other aromatic amino acids (e.g., tyrosine) indicatesthat binding is due, at least in part, to intercalation of W108 intodsDNA. As such, this domain also should bind dsRNA. Importantly, bindingof nucleic acid molecules by the peptide does not seem to have anydetectable sequence preference.

The present invention also relates to other peptide domains that sharethe desirable characteristics as described above and exemplified by theNuc peptide. Other non-sequence-specific DNA binding proteins or domainsthat can be used as a transfection reagent as disclosed herein include,for example, the region of amino acid residues 22 to 44 of mouseintermediate filament (IF) protein vimentin [Shoeman, 1999 #68], whereinDNA binding can be mediated by intercalation into dsDNA. Other suchdomains include those from HU proteins [Grove, 2001 #69], HMG1/2 [Saito,1999 #70], DNA topoisomerase I, and the like, which exhibit the abovedescribed properties desired of a transfection reagent of the invention.

The present invention also provides composition that includes aDNA-binding peptide as disclosed herein alone, or in combination with orconjugated to other peptide domains. The selected or designed DNAbinding domain can be linked, for example, to a second functional domainthat can facilitate movement of a peptide-DNA complex across abiological membrane. As such, the peptide transfection reagent (e.g.,Nuc as set forth in SEQ ID NO:1) can be operatively linked and/oroperatively associated with a second (or more) heterologous polypeptide.The term “operatively linked” or “operatively associated” is used hereinwith respect to two or more molecules that share a covalent ornon-covalent interaction, wherein each molecule maintains all or most ofa function that the molecule exhibits alone. As such, a nucleotidesequence encoding a first peptide, e.g., Nuc, can be operatively linkedto a nucleotide sequence encoding a heterologous peptide, e.g., acellular localization domain, wherein, upon expression, the nucleotidesequences are in frame and can be expressed either as a linked fusionprotein or as two independent peptides that can associate via anon-covalent interaction. Similarly, a first peptide and a secondpeptide or other molecule can be combined, for example, in a reactionmixture, such that a covalent bond or non-covalent interaction can beformed linking or associating, respectively, the two peptides, whereineach component in the complex maintains a desired functioncharacteristic of the component in a non-complexed form. It should berecognized that a nucleic acid molecule complexed with a peptidetransfection reagent of the invention also can be considered tooperatively associated because, in such a complex, the peptidetransfection reagent maintains its function of facilitating uptake ofthe nucleic acid into a cell and the nucleic acid molecule maintains itsfunction of encoding an RNA and, if appropriate, polypeptide. As such,operatively associated molecules as disclosed herein generally arestable when exposed to physiological conditions as occur, for example,in a transfection medium, a cell culture medium, or in a cell orcellular compartment.

A heterologous polypeptide operatively linked or operatively associatedwith a peptide transfection reagent of the invention can be anypolypeptide that is not linked or associated with the peptide in nature.In one embodiment, the heterologous polypeptide is a cell localizationdomain, which can facilitate transport of the operatively linked peptidetransfection reagent and any nucleic acid molecule complexed therewith,to a particular compartment of a cell. Cell localization domains arewell known in the art and include, for example, a plasma membranelocalization domain, a nuclear localization signal, a mitochondrialmembrane localization signal, an endoplasmic reticulum localizationsignal, or the like, or a PTD such as the cationic humanimmunodeficiency virus (HIV) TAT PTD or the non-charged HIV gp41 PTD,each of which can facilitate translocation of a peptide linked theretointo a cell (see Schwarze et al., Science 285:1569-1572, 1999; Derossiet al., J. Biol. Chem. 271:18188, 1996; Hancock et al., EMBO J.10:4033-4039, 1991; Buss et al., Mol. Cell. Biol. 8:3960-3963, 1988;U.S. Pat. No. 5,776,689; Morris et al., Nucl. Acids Res. 25:2703-2736,1997; Morris et al Nucl. Acids Res. 27:3510-3517, 1999; each of which isincorporated herein by reference).

A nuclear localization signal (NLS) facilitates translocation of anucleic acid molecule, for example, a polydeoxyribonucleic acid moleculethat is transcribed by RNA polymerase III and encodes an siRNA, into thenucleus of a eukaryotic cell. Traditionally, it was believed that DNA inthe cytoplasm moves by diffusion, and that about 1 in 3,000 moleculesenter the nucleus through the nuclear pores (Zanta et al., Proc. Natl.Acad. Sci., USA 96: 91-96, 1999); cell division provides the bestopportunity for transfected DNA to be enveloped inside the nucleus.Recently, real-time multiple particle tracking revealed that PEI-DNAnanocomplexes can move towards the nucleus by motor protein-driventransport (Suh et al., Proc. Natl. Acad. Sci., USA 100: 3878-3882,2003), suggesting that, in addition to random movement, DNA moleculesalso are actively transported to the proximity of the nuclear membraneby a network involving microtubules. In the presence of microtubuledepolymerizing agent nocodazole or vinblastine, the nucleus-boundmovement of DNA was significantly hindered (Coonrod et al., Gene Ther.4: 1313-1321, 1997;Suh et al., supra, 2003). In addition, an NLS canincrease transfection efficiency as an additive to lipofection orcationic polymer complexes (Branden et al., Nat. Biotechnol. 17:784-787,1999). In another case, multiple copies of SV40 NLS were used directlyas transfection agent (Ritter et al., J. Mol. Med. 81:708-717, 2003,apparently through static interaction between the positively chargedpeptide and DNA. Whether NLS peptides already bound to DNA also canassociate with the nuclear pore complex for nuclear entry is unclear.

An NLS conveniently can be included as a component of the disclosedpeptide transfection agents However, most of the well-defined NLSpeptides are cationic (review by Nakielny and Dreyfuss, Cell 99:677-690,1999), and, therefore, may irreversibly bind DNA and adversely affecttransfection. In such a case, non-cationic NLS peptides such as two suchsequences present in human DNA topoisomerase I (Mo et al., J. Biol.Chem. 275: 41107-41107, 2000) can be used in the present compositions.One of the topoisomerase I NLS peptides is an acidic amino acids-rich,29 residue peptide, and the other has 2 neutral, 2 acidic, and only 3basic amino acids in a 7 residue sequence. Both NLS peptides displayedstrong NLS activity in natural and reporter proteins. As disclosedherein, the 7 residue topoisomerase I NLS peptide (KKIKTED; SEQ ID NO:8)does not irreversibly condense DNA and, in view of its short size, canbe conveniently synthesized for inclusion in a composition of theinvention.

In another embodiment, a heterologous peptide operatively linked to apeptide transfection reagent facilitates disruption of the nucleicacid-peptide complex, thereby releasing the nucleic acid from all or apart of the peptide transfection reagent. Such peptides are exemplifiedherein by peptides that comprise an endoprotease recognition site and bypeptides having endosomolytic activity. There are several families ofendoproteases that are abundant in the cytoplasmic compartments withpeptide cutting abilities. Such proteases can be used to advantage inorder to release the nucleic acid from bound peptides inside the cell. Asimilar strategy was utilized for plasmid transfection with cationicpeptides, although the specific effects of the protease site was notillustrated (Haines et al., Gene Ther. 8:99-110, 2001).

An endoprotease recognition site useful in a composition of theinvention is selected based on the intracellular compartment(s) in whichthe complexes pass through or localize (e.g., endosomes, endoplasmicreticulum, and Golgi body), or on the presence of proteases expressionin target cells (e.g., caspases in cells that are subject to apoptosis).For example, the cleavage site GGFLGF (SEQ ID NO:6) of the endosomalendoprotease, cathepsin D, can be placed between a PTD (e.g., SEQ IDNO:2 or SEQ ID NO:5) or other peptide component of the complex, and theNuc (SEQ ID NO:1) peptide, wherein, upon uptake of the complex via anendosome-mediated pathway, the endosomal cathepsin D cleaves andreleases a portion of the peptide from the complex. The efficacy of sucha method can be confirmed, for example, by labeling the nucleic acidmolecule and the amino end of the peptide with different fluorescentdyes to observe their localization inside cells in a time course.Peptides with or without the endoprotease cleavage site can be comparedusing a fluorescent dye assay, wherein detecting separation of thepeptide and DNA after a certain time lapse only when the peptidecontains the cleavage site confirms that the peptide is cleaved.

Whether transfection of particular cell types involves endocytosis canbe examined by comparing transfection at lower temperature, and/or inthe presence of endocytosis inhibiting agents such as cytochalasin B andbalfilmycin A. Upon confirming that the transfection process involvesendocytosis, an endosomolytic agent can be included in the nucleicacid-peptide complex. Such endosomolytic peptides are exemplified byINF7 (GLFEAIEGFIENGWEGMIDGWYG; SEQ ID NO:7; Ritter et al. 2003, J. Mol.Med. 81, 708-17; Plank et al. 2002, J. Biol. Chem. 277, 2437-2443, eachof which is incorporated herein by reference), and by mellitin (Ogris etal., J. Biol. Chem. 276: 47550-47555, 2001, which is incorporated hereinby reference). INF7, for example, is a fusogenic peptide from influenzavirus. INF7 can be operatively linked to another peptide in the complex(e.g., via a disulfide bond or by expression as a fusion protein), orcan be operatively associated with the complex (e.g., via a hydrophobicinteraction). In another embodiment, the endosomolytic agent is anon-peptide agents such as chloroquine, which can be operatively linkedto or associated with the complex.

A detectable label, which facilitates identification of a composition ofthe invention or of a sample or cell containing the composition, alsocan be operatively linked to a peptide transfection reagent, or peptidelinked thereto. The detectable label can be a peptide, polypeptide, orchemical or small organic or inorganic molecule that can be convenientlydetected. For example, a detectable label can be a molecule such as abiotin, which can be detected using avidin or streptavidin; afluorescent compound (e.g., Cy3, Cy5, Fam, fluorescein, or rhodamine); aradionuclide (e.g., sulfur-35, technicium-99, phosphorus-32, ortritium); a paramagnetic spin label (e.g., carbon-13); a bioluminescentsuch as luciferin; an enzyme such as alkaline phosphatase; or achemiluminescent compound. Methods of operatively linking a detectablelabel or other moiety to a nucleotide sequence are well known in the art(see, for example, Hermanson, “Bioconjugate Techniques”(Academic Press1996), which is incorporated herein by reference). In addition toproviding a means, for example, to detect a cell containing the peptidetransfection reagent, a detectable label or other moiety also can beused to isolate such a cell. For example, where the fusion proteinincludes an operatively linked fluorescent compound, cells containingthe peptide transfection reagent and, therefore, that contain a nucleicacid molecule complexed with the reagent, can be isolated from cellsthat do not contain the peptide/nucleic acid complex by a methods suchas fluorescent activated cell sorting (FACS). Similarly, where thedetectable label is a peptide tag such as a myc epitope, FLAG epitope,or the like, an antibody or other binding partner specific for the tag,which itself can be labeled, can be used to isolate or otherwiseidentify a cell containing the peptide/nucleic acid complex.

In one embodiment, the nine residue Nun C-terminal peptide (Nuc; SEQ IDNO:1) was fused to a 11 residue TAT peptide (SEQ ID NO:2), wherein thepeptides were separated by a single glycine residue spacer. As disclosedherein, the fusion protein (designated TAN; SEQ ID NO:3) bound to doublestranded DNA as a multimer, with an apparent dissociation constant ofabout 1×10⁻⁵ M to 1×10⁻⁴ M. When binding to plasmid DNA, the peptideappears to compact the DNA molecule into a more mobile form, as itmigrates faster on gel. This result contrasts with that observed forpreviously used peptides having DNA-compacting ability, which caused thecomplexed DNA to stay in loading wells. A smeared, but distinguishablegroup of complexes were observed when TAN bound to short linear dsDNAgenerated by PCR. These DNA-binding characteristics may have a role inthe ability of Nuc-containing peptides to increase transfectionefficiency.

When TAN was mixed with plasmid DNA in a simple water-only reaction,then applied to cultured cells, a few cells expressed the reporter genecarried on the plasmid, indicating that the peptide facilitatestransfection and, therefore, acts as a transfection reagent.Accordingly, the invention provides a transfection reagent, compositionsthat include the transfection reagent, and transfection procedureutilizing the transfection reagent.

Remarkably, divalent calcium ions (Ca⁺⁺) supplied in the transfectionreaction using CaCl₂ resulted in transfection of nearly all of the cellsin the culture. When green fluorescent protein (GFP) was used asreporter gene, the transfected cells appeared to express the reportergene to a higher level as compared to cells transfected using othermethods. Another advantage of the reagents and methods of the inventionis that a time course study revealed that the transfection complex toform quickly (after about only one minute), thus providing a very fastprocedure. Additionally, it took less time for the transfected genes tobe expressed using the peptides and methods of the invention as comparedto liposome or other commercial transfection systems. The treated cellsdid not show arrest of growth or cell death that typically is observedafter treatment with several liposome or PEI transfection reagents,indicating that the transfection reagents of the invention exhibit lowtoxicity.

Agreeing with the result of using calcium ion for Nuc-mediatedtransfection, calcium ion stabilized the peptide-DNA complex in aband-shift assay. The dramatic enhancement of transfection by calciumion can be due, at least in part, to its effect on the complex formationbetween multiple peptide and DNA. Of note, no phosphate was added forthis effect, thus demonstrating that a calcium phosphate precipitatedoes not have a significant role in the observed results, though thepossibility that calcium ion somehow helps opening up the cell membranecannot be ruled out.

The invention also relates to further usage of the peptides of theinvention in combination with other transfection reagent. A several foldincrease of transfection efficiency was consistently observed when thepeptide transfection reagent was bound to DNA first, then mixed withliposome-based transfection reagent.

The invention further relates to compositions including a DNA-bindingpeptide domain transfection reagent operatively linked to (oroperatively associated with) one or more (e.g., 2, 3, 4, etc.) otherpeptide domains and/or other molecules for transfection into particularcell types or in animals or humans. Such domains include, but are notlimited, to PTD, NLS, endoprotease, and endosomolytic sequences ormolecules, as discussed above, as well as natural or artificial domainsthat can destabilize or pass through a biological membrane. PTDs havebeen derived from HIV TAT protein (TAT) [Becker-Hapak, 2001 #39], thehomeodomain of Drosophila transcription factor Antennapedia, the HSVprotein VP22, basic fibroblast growth factor, and HIV gp41, etc. Othermembrane-active peptide include melittin and the like. A few peptidesalso can posses similar properties by design, including, for example,MPG, Pep-1, and oligomers of L-arginine and D-arginine, lysine, andhistidine. TAT has been used to deliver large fusion proteins intovarious cells and adult animals, and also crosses the blood-brainbarrier effectively [Schwarze, 1999 #10].

Also provided are kits, which include at least a transfection reagentcomprising the Nuc peptide for transfecting eukaryotic cells. A kit ofthe invention also can contain one or more additional reagents usefulfor practicing or monitoring a transfection reaction including, forexample, a calcium ion source (e.g., CaCl₂), a standard nucleic acidmolecule, or complex thereof, useful for monitoring transfectionefficiency, buffers, or other such materials typically used in atransfection reaction. In addition, the kit can contain one or moreother peptides, as desired, for example, a TAT peptide, a nuclearlocalization signal, and the like, which can be separate component ofthe kit, thus providing a means to select and complex the Nuc peptide tothe other peptide, or can be in the form of a fusion peptide with theNuc peptide.

The methods and compositions of the invention can be useful fortransfecting cells in vitro, including cells adapted to culture (e.g.,cell lines or panels of cells that have adapted to culture) or cells exvivo (i.e., cells that have been removed from a subject such as a humansubject for the purpose of manipulating (transfecting) the cells inculture, expanding (if desired) the manipulated cells, andre-administering the cells back into the same or a different, generallya haplotype-matched, subject). In addition, the methods and compositionsof the invention are useful for introducing a nucleic acid molecule intocells of a subject in vivo, thus providing methods for performing animalstudies, including for example, developing transgenic animals, which canexpress a desired gene product or provide a desired animal model of adisease, particularly a human disease, as well as providing methods ofhuman gene therapy. It is known that most of the widely availablecationic lipids, including, for example, LIPOFECTAMINE transfectionreagent and DC-cholesterol, have a very poor ability to enhance DNAexpression above the baseline level with naked DNA in animals [Felgner,1995 #157; Ferrari, 1999 #158]. From studies of TDP and NLS functions,it is clear that peptides possess and extremely potent ability to bringmacromolecules into cells and organs of animals. As such, peptides asdisclosed herein, in combination with a peptide transfection reagent ofthe invention and, if desired, other reagents, can be used for enhancinggene transfer by non-viral means.

The methods of the present invention can be conveniently adapted to ahigh throughput format, thereby allowing for two or more transfectionreactions to be performed in parallel. Accordingly, the presentinvention also provides methods of performing a plurality (i.e., 2 ormore) transfection reactions in a high throughput format, including, forexample, on a solid support, wherein individual and discrete reactionscan be performed. The solid support can be any substrate typically usedfor performing a high throughput assay (e.g., a silicon wafer, a glassslide, or a bead), and the samples can, but need not, be arranged in anarray, which can be an addressable array. Microarray technology has beenapplied to many areas of biomedical analysis, including for monitoringgene expression, genotyping single nucleotide polymorphisms (SNP), andsequencing.

The principle of microarrays of cells was recently established (Ziauddinand Sabatini, Nature 411:107-110, 2001), thus allowing expression of adefined cDNA in a cluster of cells grown on a slide upon which hundredsof cDNAs in expression vectors were spotted. The success of this methodsdepends on the cDNA molecules being “printed” on the slides in such away that they can be moved away from the solid support (i.e., into acell), and the cDNA molecules being in form suitable for internalizationby cells. In the original methods, cDNA was mixed with a gelatinsolution before spotting onto the slide. After drying, the cDNA spotswere exposed to transfection reagents, then the slides were placed in aculture dish and overlaid with adherent mammalian cells in medium.Because of the reversed order-of-addition of DNA molecules and cells,this process is referred to as “reverse transfection”.

Cell arrays have been successfully applied to RNAi analysis based on thereverse transfection principles, and using the gelatin method asoriginally described, or using sucrose and MATRIGEL matrix for embeddingsiRNAs complexed with lipofection agents before spotting (Kumar et al.,Genome Res. 13:2333-2340, 2003; Mousses et at., Genome Res.13:2341-2347, 2003, each of which is incorporated herein by reference).Unfortunately, these embedding methods have two major shortcomings.First, DNA spotted in gelatin-like solutions is not highly confined and,therefore, are not conveniently adaptable for generating high densitymicroarrays and, generally, are limited to the use of a few hundredssiRNA spots per slide, which is far below the upper limit set by theattainable cell density (Ziauddin and Sabatini, supra, 2001). Forcomparison, the hybridization DNA chips can be printed at 1 millionfeatures per chip (e.g., a GeneChip™ microarray; Affymetrix). Thislimitation prevents the use of the current RNAi chips for genome-wide orrandom RNAi library screening. The second limitation of the reversetransfection method relates to the transfection efficiency. A typicaltransfection reagent seldom gives 100% efficiency under normal tissueculture conditions, and is much lower when the DNA is embedded in asemisolid carrier protein layer. Unfortunately, a high transfectionefficiency generally is required to obtain meaningful down-regulation byRNAi, as compared to that required to observe positive gene expressionby cDNA.

As disclosed herein, linear DNA cassettes encoding siRNAs can be used togenerate high density arrays. Instead of semisolid embedding, the linearcassette molecules is immobilized by one end onto a slide, similar toany chip containing hybridizing oligonucleotides. For the immobilizedDNA to be internalized by cell, it is printed via a transmembrane domain(TMD) peptide with an intramembrane cleaving protease site (I-CliPs).I-CliPs are a rapidly expending family of proteases and peptidases thatunexpectedly hydrolyze substrate proteins or peptides within thehydrophobic environment of membrane lipid bilayers Wolfe and Selkoe,Science 296:2156-2157, 2002). The presenilin 1 and presenilin 2 I-CliPsare selected for exemplifying the present methods because they arenearly ubiquitously expressed, and can cut peptides within the cellmembrane (other I-CliPs cleave within ER or Golgi membranes) so as torelease the cytosolic portion of the substrate into the cytoplasm or, insome cases, into the nucleus Lee et al., J. Neurosci. 16: 7513-7525,1996). In the exemplified method, the TMD peptide is synthesized with abiotin group at one end, linked to the DNA at the other. Thebiotinylated molecule can be spotted to streptavidin-coated chips at ahigh density commensurate to the optimum growth density of the cells.The TMD can be cleaved by I-CliPs once the DNA is uplifted into a cell.

A TMD sequence can be prepared using standard procedures, and anN-terminal biotin can be added using biotin-N-hydroxysuccinimide ester.A linear cassette of about 125 bp encoding an siRNA can be generated byoligonucleotide synthesis, which each strand synthesized to contain a 5′amino group and conjugated to a cysteine residue on either the TMD orthe PTD-containing peptide. Crosslinking can be achieved usingSulfo-SMCC reagent. Peptide-conjugated sense and antisense strands arehybridized prior to spotting onto streptavidin-coated slides.Alternatively, the linear nucleic acid cassettes can be conjugated onlyto the TMD peptide (and not the PTD peptide), then, after spotting, thechip is soaked in solutions containing the nucleic acid-peptide complex.The cells then can be overlaid. Such methods for preparing themicroarrays, including, for example, spotting the nucleic acid-peptidecomplex onto the substrate and contacting the arrays with cells, can beperformed manually, or can be partially or fully automated, as canfurther steps of examining the transfected cells for expression of theintroduced nucleic acid molecule.

The following examples are provided to illustrate aspects of the presentinvention.

EXAMPLES

There are many potential benefits to using peptide as transfectionreagent, including, for example, they are relatively easy tomanufacture, store and use; have low toxicity; have low cost; areflexible to use alone, or in combination with other reagents; and areeasy to modify and reformulate. Such peptide transfection reagents areexemplified herein by a fusion protein, which is composed of at leasttwo functional components, including a DNA binding/compacting module anda biological membrane affinity/passing module. The effectiveness of thedisclosed compositions is demonstrated using the C-terminal domain ofthe Nun protein (“Nuc”) as the DNA-binding domain, and the HIV TAT asthe membrane transduction domain. Although TAT was used because it is awell-studied targeting peptide, other peptides having similar membranetranslocating activity similarly can be used. For example, TAT carries astrong positive charge and, therefore, can bind to negatively chargedmolecules surrounding cells. As such, signal peptides that are notcationic also can be used as a component of a fusion protein includingNuc, such that non-specific binding is reduced. One such example is theNLS of amino acids 117-146 of human DNA topoisomerase I and itscounterparts in mouse, hamster, chicken, frog, and other species (Mo etal., J. Biol. Chem. 52:41107-13. 2000). This domain is negativelycharged and, therefore, will not bind to many serum proteins andpolyglycans. It can be further modified to be neutral to further avoidnon-specific binding.

Example 1 demonstrates that a fusion peptide between Nuc and TAT iscapable of entering cells. Example 2 illustrates that the fusionpeptide, TAN, can bring reporter DNA plasmid into cells as atransfection reagent. Example 3 further shows that the transfection withTAN can be greatly enhanced with the addition of low amount of calcium.Example 4 further shows that TAN can facilitate transfection in lipid-or cationic polymer-based transfection systems. Example 5 providesevidence that the TAN transfection system can be applied to manydifferent cell types, some of which are known to bedifficult-to-transfect. Example 6 is the result of a direct bindingassay between TAN and dsDNA plasmid. Example 7 shows that TAN can alsocomplex with short linear dsDNA that functions in RNAi.

Example 1 Transduction Abilities of Peptide Tan

TAN and TAT having the following sequences were synthesized by Fmocchemistry (SynPep Corp.; Dublin Calif.): TAT: YGRKKRRQRRR; (SEQ ID NO:2)and TAN: YGRKKRRQRRRGQRNPNKKWS. (SEQ ID NO:3)

NHS-Fluorescein was purchased from Pierce Chemical Co. (Rockford Ill.).To label the peptides with the fluorescent dye, 500 μg of peptide wasmixed with 250 μg of NHS-Fluorescein in DMSO, incubated for 2 hours onice, purified by a PD10 column and eluted in 0.75 ml fractions of PBSbuffer. The labeling and purification of the peptide was examined byHPLC. Fractions 2 and 3 had the majority of the labeled peptides.

293T (human embryonic kidney) cells were grown in Dulbecco's modifiedEagle's medium (Life Technologies, Rockville, Md.) supplemented with 10%FBS, 100 units/ml penicillin, and 100 μg/ml of streptomycin in a 37° C.CO₂ incubator. Cells were regularly passed to maintain growth.Twenty-four hours before transfection, cells were trypsinized and platedon 96-well plates (100 μl/well) in the above medium without theantibiotics. Ten μl of fraction 2 was added to 293T cells at 15%confluency in serum-containing medium. Fluorescence microscopy wasperformed at 12 hours, after changing medium and washing cells.

Fluorescence was observed throughout the transducted cells. Examinationof a single cell revealed that the cell divided by 24 hours, and thatthe resulting two daughter cells also showed evenly distributedfluorescent light, but of lower intensity due to dilution. Thisexperiment demonstrates that the peptide encompassing Nuc and a knownsignal peptide effectively cross the cell membrane of eukaryotic cells.

Example 2 Transfection Abilities of Peptide Tan

TAN (SEQ ID NO:3) at 0.1 to 5 μg per 96-well transfection was mixed with0.1 μg plasmid pEGFP-N1 (BD ClonTech) for 5 min, diluted in 40 μl DMEM,then added to cells. Fluorescence microscopy was performed at differenttime points to observe the expression of GFP as the indication oftransfection event. Note that by using GFP as reporter, the totaltransfection efficiency was underestimated due to certain percentage ofcells would not express the GFP to levels high enough for detection. Aset of photographs demonstrated the dose effect of peptide TAN incombination with Ca⁺⁺ on transfection, indicating that there is aspecific effect by TAN under a given Ca⁺⁺ concentration and vise versa.

Example 3 Transfection Abilities of Peptide Tan in Combination withCalcium Ion

Different concentrations of TAN (SEQ ID NO:3) and CaCl₂ were tested incombination for transfection. TAT (SEQ ID NO:1) and another peptide, HT31 (a widely used PKA-anchor interaction interruption peptide) wereincluded as controls. Generally, experiments were performed similarly asin the above examples except that peptide was added to DNA first,followed by addition of CaCl_(2.) Nonetheless, an order-of-additionexperiment indicated that adding peptide, CaCl_(2,) or DNA in any orderresult in similar transfection efficiency. As control, TAT together withCaCl₂ caused cells to lyse, while the HT31 showed no effect.

As little as 0.1 μg of peptide in the presence of 15 mM of calciumresulted in high transfection efficiency. TAN was not examined atamounts less than 0.1 μg, but may nevertheless mediate transfection tosimilar or higher levels. The “charge ratio”, defined as the ratiobetween the total number of the positive charges on the peptide and thetotal negative charges on the DNA, was close to 1:1. This ratio is lowerthan typical cationic peptide-mediated transfections, agreeing with thenotion that the binding of this peptide is not mainly throughelectrostatic interaction. The amount of DNA used to achieve thedemonstrated levels of transfection using our peptide transfectionsystem is also lower than those normally suggested for commercialtransfection reagents.

The effects of calcium ion was dose-dependent, with a bell curve peakingat about 30 mM calcium with 0.5 μg TAN peptide. Calcium phosphateprecipitation is one of the oldest methods of transfection. It ispossible that the effect of Ca⁺⁺ on transfection in the TAN system wasdue to the interaction between Ca⁺⁺ and phosphate presented by themedium. To test this, transfection experiments were performed with TANin PBS or Mono-Q™ column desalted H₂O. The inclusion of phosphate in thereaction actually decreased transfection efficiency, thus demonstratingthat the effect of combining TAN and calcium ion was not enhanced by thepresence of phosphate, and indicating that TAN likely acts by amechanism different from calcium phosphate precipitation.

This result indicates that calcium in its free ionic state, instead ofas a phosphate precipitate, is the functional component in enhancing theTAN-mediated transfection. In addition, with different amounts of thepeptide, the optimum concentration of calcium ion can change, suggestinga defined relationship between the two factors in forming celltransfecting DNA complexes. Furthermore, in forming DNA precipitates bya typical calcium phosphate transfection protocol, the concentration ofcalcium is 0.122 M, whereas in the reactions exemplified herein, theconcentration was about 10-fold lower. Higher calcium ion concentrationssometimes resulted in severe cell toxicity.

MgCl₂, NaCl and other salts were included as control and showed noeffect on enhancing TAN-mediated transfection, indicating specificity ofcalcium ion. A time course was performed in order to find out theoptimum incubation time period of forming TAN-DNA-Ca⁺⁺ complexes. Thereaction appeared to reach the highest transfection effect quickly(within 1 min, and peaking by 5 min), which is advantageous fortransfection, suggesting a preferable kinetics for most applications incomparison with other methods.

Example 4 Transfection Enhancing Effects of Peptide Tan in Combinationwith other Transfection Reagents

To determine whether TAN increased transfection efficiency when used asan additive to other transfection systems, several commercialtransfection reagents were used accordingly to the manufacturers'protocols, but with TAN (SEQ ID NO:3) or control peptides added to theDNA first. Preliminary data indicated that TAN increased transfectionefficiency of various transfection reagents under different conditions.Five μg of TAN (SEQ ID NO:3) or TAT (SEQ ID NO:2) was added to DNAbefore performing transfection with TransIt-Oligo™ reagent (Mirus;Madison Wis.). After incubating peptide with DNA for 5 min, 0.5 μl ofTransIt-Oligo™ reagent diluted in 10 μl of DMEM was added and themixture was further incubated for 15 min before overlaying onto 293Tcells as described above. Several fold higher GFP expression wasconsistently observed with TAN than with control peptides. These resultsdemonstrate that TAN (SEQ ID NO:3) can increase the transfection abilityof other transfection systems, for example, by accompanying DNA intocells and nuclei.

Example 5 Transfection of Difficult-To-Transfect Cells Using the TanTransfection System

In order to test whether transfection by the novel peptide could beapplied to other cell types, including cells that are known to bedifficult to transfect with existing methods, several different celllines were examined using the procedures described above. GFP expressionwas enhanced by TAN (SEQ ID NO:3) in a TransIt-Oligo™ reagent-mediatedtransfection. In this case, although the total number of GFP-positivecells did not appear to increase in the presence of TAN, the cells thatwere transfected using TAN were much brighter as compared to thosetransfected without TAN. The experiments were done in 24-well plate with3T3-L1 cells 15 days post-induction, a time point when transfection isnormally extremely difficult. Note that the amount of DNA used was alsovery limited (0.2 μg in reaction 1-3, 0.5 μg in reaction 4).

Transfection was examined with monkey kidney cell Vero-E6 (ATCC) cellline, which is routinely used for human disease related viral infection,e.g. SARS, as a drug testing model system. Vero-E6 cells with limitedpassages from original ATCC stock culture were grown in 96-well plate atabout 30% confluency. Transfections were performed with 0.1 μg ofplasmid pEFGP-N1 as described above. Two doses of TransIt-Oligo™ reagentwere tested, but neither provided good transfection under thisconditions. However, with increasing amount of TAN (SEQ ID NO:3), thetransfected cells were more prominent. In parallel control experiments,TAT (SEQ ID NO:2) did not show such enhancement.

Example 6 Tan Compacts Plasmid DNA and Calcium Facilitates ComplexFormation

In order to examine whether TAN could directly interact with thetransfected plasmid DNA as predicted by the transfection results,band-shift assays were performed. Different amount of TAN (SEQ ID NO:3)dissolved in H₂O was mixed with 0.5 μg of plasmid with or without 15 mMCaCl₂, incubated for 5 min, followed by addition of 2 μl of DNA loadingdye (30% glycerol), and loaded to 0.7% agarose gel. The EB-containinggel was run for about 45 min at 120 volts before the picture taken underUV lights.

Two types of changes were observed as result of TAN: 1) some plasmidstayed in the wells, as seen with almost all published reports on suchpeptide band-shift assays, presumably due to the aggregation of DNAinduced by peptide binding; and 2) a certain amount of plasmid migratedmore quickly, as a distinct band, than even the supercoiled plasmid,suggesting they were compacted but not in any large aggregates.Significantly, in the presence of calcium ion, the complex became moreintense, while all other bands decreased. These results indicate thatcalcium ion can stabilize the TAN-DNA complex, thus reducingaggregation. Taken together with the transfection data, these resultsindicate that calcium ion can affect TAN-mediated transfection, at leastin part, at the step of complex formation.

Example 7 Tan Binds Linear Cassettes of Sirna-expressing DSDNA

The ability of TAN (SEQ ID NO:3) to bind short linear dsDNA also wasexamined. The DNA tested was an approximately 300 bp PCR product that isused to express small interfering RNAs (siRNAs). The experiments weredone similarly to those described in Example 6, except that thereactions were allowed to proceed to 30 min and the gel was 2%. TAN (SEQID NO:3) was found to bind to the linear dsDNA in a dose-dependentmanner, with a Kd of approximately 1.5×10⁻⁴M⁻¹ which is in the samerange as was observed for the double stranded plasmid DNA.Interestingly, the complexes formed between DNA and TAN migrated moreslowly than the free DNA, which is different from those between plasmidand TAN. This difference may be due to a difference in the relativeamount of DNA in each type of complex or to the structural constrains oflinear versus circular DNA. As controls, peptide HT31 did not bind theDNA at similar concentrations. TAT (SEQ ID NO:2) also did not bind atthe lower concentrations, and quickly precipitated DNA as concentrationincreased slightly. The TAN peptide also enhanced the RNAi effects whenused in transfection aimed at causing gene silencing by thesiRNA-expressing cassettes. Because these cassettes express siRNAs underthe control of Pol III promoter as apposed to Pol II in GFP expressionunit, it is therefore likely that peptide assisted transfection (PAT) isindependent of promoter used, as expected.

References Cited:

Each of the following references is incorporated herein by reference:

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Although the invention has been described with reference to the aboveexample, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

1. A peptide having an amino acid sequence consisting of QRNPNKKWS (SEQID NO:1).
 2. The peptide of claim 1, which comprises a peptide fragmentof a Nun polypeptide.
 3. A fusion protein, comprising the peptide of SEQID NO:1 operatively linked to a heterologous peptide.
 4. The fusionprotein of claim 3, wherein the heterologous peptide comprises acellular localization domain.
 5. The fusion protein of claim 4, whereinthe cellular localization domain comprises a nuclear localizationsignal.
 6. The fusion protein of claim 4, wherein the nuclearlocalization signal comprises SEQ ID NO:4 or SEQ ID NO:8.
 7. The fusionprotein of claim 4, wherein the cellular localization signal comprises ahuman immunodeficiency virus (HIV) TAT peptide or an HIV gp41 peptide.8. The fusion protein of claim 7, wherein the HIV TAT peptide comprisesan amino acid sequence as set forth in SEQ ID NO:2, and wherein the HIVgp41 peptide comprises an amino acid sequence as set forth in SEQ IDNO:5.
 9. The fusion protein of claim 7, further comprising a nuclearlocalization signal.
 10. The fusion protein of claim 3, which has anamino acid sequence as set forth in SEQ ID NO:3.
 11. The fusion proteinof claim 3, which further comprises an endoprotease recognition site, anendosomolytic peptide, or a combination thereof.
 12. A composition,comprising the peptide of claim 1 and a heterologous polypeptide.
 13. Acomposition, comprising the peptide of claim 1 and a nucleic acidmolecule.
 14. The composition of claim 13, wherein the nucleic acidmolecule comprises a double stranded nucleic acid molecule.
 15. Thecomposition of claim 13, wherein the nucleic acid molecule comprises DNAor RNA.
 16. A composition, comprising the peptide of claim 1 anddivalent calcium ions.
 17. The composition of claim 13, furthercomprising divalent calcium ions.
 18. A kit, comprising the peptide ofclaim
 1. 19. The kit of claim 18, wherein the peptide of claim 1 isoperatively associated with a heterologous polypeptide.
 20. The kit ofclaim 19, wherein the peptide of claim 1 and the heterologouspolypeptide comprise a fusion protein.
 21. The kit of claim 20, whereinthe fusion protein comprises, in operative linkage, the peptide of claim1 operatively linked to a peptide having an amino acid sequencecomprising SEQ ID NO:2 or SEQ ID NO:5.
 22. The kit of claim 21, whereinthe fusion protein has an amino acid sequence as set forth in SEQ IDNO:3.
 23. The kit of claim 18, further comprising at least oneheterologous polypeptide, which can be operatively linked to oroperatively associated with the peptide of claim
 1. 24. The kit of claim23, which comprises a plurality of heterologous polypeptides, wherein atleast two heterologous polypeptides of the plurality are different. 25.The kit of claim 18, further comprising a transfection reagent.
 26. Thekit of claim 25, wherein the transfection reagent comprises divalentcalcium ions.
 27. A solid substrate, which comprises at least onepeptide of claim
 1. 28. The solid substrate of claim 27, which comprisesa plurality of peptides of claim
 1. 29. The solid substrate of claim 28,wherein the peptides of the plurality are in array.
 30. The solidsubstrate of claim 28, wherein the peptide of claim 1 comprises acomplex with a nucleic acid molecule.
 31. A method of transfecting acell, comprising contacting the cell with the peptide of claim 1 and anucleic acid molecule under conditions sufficient from celltransfection.
 32. The method of claim 31, wherein said conditionscomprise further contacting the cell with divalent calcium ions.
 33. Themethod of claim 31, wherein the peptide of claim 1 further comprises anoperatively associated heterologous polypeptide.
 34. The method of claim31, wherein the peptide of claim 1 is attached to a solid substrate. 35.A method of transfecting a cell, comprising contacting the cell with thefusion protein of claim 3 and a nucleic acid molecule under conditionssufficient from cell transfection.
 36. The method of claim 35, whereinthe heterologous polypeptide comprises an HIV TAT peptide or an HIV gp41peptide.
 37. The method of claim 36, wherein the fusion protein furthercomprises a nuclear localization signal.
 38. The method of claim 35,wherein the fusion protein has an amino acid sequence as set forth inSEQ ID NO:3.
 39. The method of claim 35, wherein said conditionscomprise further contacting the cell with divalent calcium ions.